Axons must withstand mechanical forces, including tension, torsion, and compression. Spectrins and actin form a periodic cytoskeleton proposed to protect axons against these forces. However, because spectrins also participate in assembly of axon initial segments (AISs) and nodes of Ranvier, it is difficult to uncouple their roles in maintaining axon integrity from their functions at AIS and nodes. To overcome this problem and to determine the importance of spectrin cytoskeletons for axon integrity, we generated mice with αII spectrin-deficient peripheral sensory neurons. The axons of these neurons are very long and exposed to the mechanical forces associated with limb movement; most lack an AIS, and some are unmyelinated and have no nodes. We analyzed αII spectrin-deficient mice of both sexes and found that, in myelinated axons, αII spectrin forms a periodic cytoskeleton with βIV and βII spectrin at nodes of Ranvier and paranodes, respectively, but that loss of αII spectrin disrupts this organization. Avil-cre;Sptan1f/f mice have reduced numbers of nodes, disrupted paranodal junctions, and mislocalized Kv1 K+ channels. We show that the density of nodal βIV spectrin is constant among axons, but the density of nodal αII spectrin increases with axon diameter. Remarkably, Avil-cre;Sptan1f/f mice have intact nociception and small-diameter axons, but severe ataxia due to preferential degeneration of large-diameter myelinated axons. Our results suggest that nodal αII spectrin helps resist the mechanical forces experienced by large-diameter axons, and that αII spectrin-dependent cytoskeletons are also required for assembly of nodes of Ranvier.SIGNIFICANCE STATEMENT A periodic axonal cytoskeleton consisting of actin and spectrin has been proposed to help axons resist the mechanical forces to which they are exposed (e.g., compression, torsion, and stretch). However, until now, no vertebrate animal model has tested the requirement of the spectrin cytoskeleton in maintenance of axon integrity. We demonstrate the role of the periodic spectrin-dependent cytoskeleton in axons and show that loss of αII spectrin from PNS axons causes preferential degeneration of large-diameter myelinated axons. We show that nodal αII spectrin is found at greater densities in large-diameter myelinated axons, suggesting that nodes are particularly vulnerable domains requiring a specialized cytoskeleton to protect against axon degeneration.

Spectrins form a submembranous cytoskeleton proposed to confer strength and flexibility to neurons and to participate in ion channel clustering at axon initial segments (AIS) and nodes of Ranvier. Neuronal spectrin cytoskeletons consist of diverse β subunits and αII spectrin. Although αII spectrin is found in neurons in both axonal and somatodendritic domains, using proteomics, biochemistry, and superresolution microscopy, we show that αII and βIV spectrin interact and form a periodic AIS cytoskeleton. To determine the role of spectrins in the nervous system, we generated Sptan1f/f mice for deletion of CNS αII spectrin. We analyzed αII spectrin-deficient mice of both sexes and found that loss of αII spectrin causes profound reductions in all β spectrins. αII spectrin-deficient mice die before 1 month of age and have disrupted AIS and many other neurological impairments including seizures, disrupted cortical lamination, and widespread neurodegeneration. These results demonstrate the importance of the spectrin cytoskeleton both at the AIS and throughout the nervous system.SIGNIFICANCE STATEMENT Spectrin cytoskeletons play diverse roles in neurons, including assembly of excitable domains such as the axon initial segment (AIS) and nodes of Ranvier. However, the molecular composition and structure of these cytoskeletons remain poorly understood. Here, we show that αII spectrin partners with βIV spectrin to form a periodic cytoskeleton at the AIS. Using a new αII spectrin conditional knock-out mouse, we show that αII spectrin is required for AIS assembly, neuronal excitability, cortical lamination, and to protect against neurodegeneration. These results demonstrate the broad importance of spectrin cytoskeletons for nervous system function and development and have important implications for nervous system injuries and diseases because disruption of the spectrin cytoskeleton is a common molecular pathology.

Paranodal axoglial junctions are critical for maintaining the segregation of axonal domains along myelinated axons; however, the proteins required to organize and maintain this structure are not fully understood. Netrin-1 and its receptor Deleted in Colorectal Cancer (DCC) are proteins enriched at paranodes that are expressed by neurons and oligodendrocytes. To identify the specific function of DCC expressed by oligodendrocytes in vivo, we selectively eliminated DCC from mature myelinating oligodendrocytes using an inducible cre regulated by the proteolipid protein promoter. We demonstrate that DCC deletion results in progressive disruption of the organization of axonal domains, myelin ultrastructure, and myelin protein composition. Conditional DCC knock-out mice develop balance and coordination deficits and exhibit decreased conduction velocity. We conclude that DCC expression by oligodendrocytes is required for the maintenance and stability of myelin in vivo, which is essential for proper signal conduction in the CNS.

In the last two decades, sensory neurons and Schwann cells in the dorsal root ganglia (DRG) were shown to express the rate-limiting enzyme of the steroid synthesis, cytochrome P450 side-chain cleavage enzyme (P450scc), as well as the key enzyme of progesterone synthesis, 3β-hydroxysteroid dehydrogenase (3β-HSD). Thus, it was well justified to consider that DRG neurons similarly are able to synthesize progesterone de novo from cholesterol. Because direct progesterone effects on axonal outgrowth in peripheral neurons have not been investigated up to now, the present study provides the first insights into the impact of exogenous progesterone on axonal outgrowth in DRG neurons. Our studies including microinjection and laser scanning microscopy demonstrate morphological changes especially in the neuronal growth cones after progesterone treatment. Furthermore, we were able to detect a distinctly enhanced motility only a few minutes after the start of progesterone treatment using time-lapse imaging. Investigation of the cytoskeletal distribution in the neuronal growth cone before, during, and after progesterone incubation revealed a rapid reorganization of actin filaments. To get a closer idea of the underlying receptor mechanisms, we further studied the expression of progesterone receptors in DRG neurons using RT-PCR and immunohistochemistry. Thus, we could demonstrate for the first time that classical progesterone receptor (PR) A and B and the recently described progesterone receptor membrane component 1 (PGRMC1) are expressed in DRG neurons. Antagonism of the classical progesterone receptors by mifepristone revealed that the observed progesterone effects are transmitted through PR-A and PR-B.

Both vagal and sacral neural crest cells contribute to the enteric nervous system in the hindgut. Because it is difficult to visualize sacral crest cells independently of vagal crest, the nature and extent of the sacral crest contribution to the enteric nervous system are not well established in rodents. To overcome this problem we generated mice in which only the fluorescent protein-labeled sacral crest are present in the terminal colon. We found that sacral crest cells were associated with extrinsic nerve fibers. We investigated the source, time of appearance, and characteristics of the extrinsic nerve fibers found in the aganglionic colon. We observed that the pelvic ganglion neurons contributed a number of extrinsic fibers that travel within the hindgut between circular and longitudinal muscles and within the submucosa and serosa. Sacral crest-derived cells along these fibers diminished in number from fetal to postnatal stages. A small number of sacral crest-derived cells were found between the muscle layers and expressed the neuronal marker Hu. We conclude that sacral crest cells enter the hindgut by advancing on extrinsic fibers and, in aganglionic preparations, they form a small number of neurons at sites normally occupied by myenteric ganglia. We also examined the colons of ganglionated preparations and found sacral crest-derived cells associated with both extrinsic nerve fibers and nascent ganglia. Extrinsic nerve fibers serve as a route of entry for both rodent and avian sacral crest into the hindgut.

This paper provides the first evidence that freshwater turtles are able to reconnect their completely transected spinal cords, leading to some degree of recovery of the motor functions lost after injury. Videographic analysis showed that some turtles (5 of 11) surviving more than 20 days after injury were able to initiate stepping locomotion. However, the stepping movements were slower than those of normal animals, and swimming patterns were not restored. Even though just 45% of the injured turtles recovered their stepping patterns, all showed axonal sprouting beyond the lesion site. Immunocytochemical and electron microscope images revealed the occurrence of regrowing axons crossing the severed region. A major contingent of the axons reconnecting the cord originated from sensory neurons lying in dorsal ganglia adjacent to the lesion site. The axons bridging the damaged region traveled on a cellular scaffold consisting of brain lipid-binding protein (BLBP)- and glial fibrillary acidic protein (GFAP)-positive cells and processes. Serotonergic varicose nerve fibers and endings were found at early stages of the healing process at the epicenter of the lesion. Interestingly, the glial scar commonly found in the damaged central nervous system of mammals was absent. In contrast, GFAP- and BLBP-positive processes were found running parallel to the main axis of the cord accompanying the crossing axons.

The largest of the cranial ganglia, the trigeminal ganglion, relays cutaneous sensations of the head to the central nervous system. Its sensory neurons have a dual origin from both ectodermal placodes and neural crest. Here, we show that the birth of neurons derived from the chick ophthalmic trigeminal placode begins prior to their ingression (HH11), as early as HH8, and considerably earlier than previously suspected (HH16). Furthermore, cells exiting the cell cycle shortly thereafter express the ophthalmic trigeminal placode marker Pax3 (HH9). At HH11, these postmitotic Pax3+ placode cells begin to express the pan-neuronal marker neurofilament while still in the ectoderm. Analysis of the ectodermal origin and distribution of these early postmitotic neurons reveals that the ophthalmic placode extends further rostrally than anticipated, contributing to neurons that reside in and make a significant contribution to the ophthalmic trigeminal nerve. These data redefine the timing and extent of neuron formation from the ophthalmic trigeminal placode.

Recent chick experiments have shown that Notch signaling plays context-dependent distinct roles in inner ear development: initially, Notch activity confers a prosensory character on groups of cells by "lateral induction"; subsequently, it is involved in the establishment of fine-graded patterns of hair cells and supporting cells by "lateral inhibition." However, the spatiotemporal pattern of Notch activation in situ during mammalian inner ear development has not been investigated. In this study, we detected the expression patterns of the activated form of Notch1 (actN1) as well as those of endogenous Notch1, Jagged1 (Jag1), and Math1. ActN1 was detected by immunohistochemistry using an antibody that specifically recognizes the processed form of the intracellular domain of Notch1 cleaved by presenilin/gamma-secretase activity. Between embryonic days (E)12.5 and E14.5, actN1 was weakly detected mainly in the medial region of cochlear epithelium, where Jag1-immunoreactivivty (IR) was also observed. Jag1-IR gradually became stronger in a more sharply defined area, finally becoming localized in supporting cells, while actN1 was detected in an overlapping area. Thus, a positive feedback loop was assumed to exist between the expression of Jag1 and actN1. In addition, actN1 started to be strongly expressed in the cells surrounding Math1-positive hair cell progenitors between E14.5 and E15.5. Strong actN1-IR continued in both a supporting cell lineage and in the greater epithelial ridge during the perinatal stage but ended by P7, suggesting that Notch1 activation may initially demarcate a prosensory region in the cochlear epithelium and then inhibit progenitor cells from becoming hair cells via classical "lateral inhibition."

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